The formation of reactive metabolites leading to specific DNA adducts and the persistence of these adducts in target tissues may be the key steps in carcinogenesis by the environmental nitrosamines, N-nitro-sopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), and N-nitrosodiethanolamine (NDELA). Metabolic and structure-activity studies indicate that alhpa-hydroxylation leads to DNA adduct formation. However, the DNA adducts resulting from activation of NPYR, NMOR, and NDELA have not been identified and the factors which influence their formation and persistence in target tissues are unknown. Therefore, the specific aims of this program are: (1) determine the structures of the DNA adducts formed upon metabolic activation NPYR, NMOR and NDELA; (2) determine the relationship between the levels or persistence of specific DNA adducts and the organospecificity of NMOR or NPYR toward rat liver and hamster trachea; and (3) determine the activities and regio-specificities toward hydroxylation of NPYR, NMOR and N-nitrosopiperidine (NPIP) of rat hepatic microsomal fractions enriched in different forms of cytochrome P-450. DNA adducts will be identified by comparison of chromatographic properties of the adducts formed in vivo from radiolabelled carcinogens with those of synthetic reference markers. Chromatographic systems developed to separate and quantify specific carcinogen DNA adducts will be employed to measure their persistence in target and non-target organs. The role of different forms of cytochrome P-450 in the activation or detoxification of nitrosamines will be assessed using biochemical assays for alpha-hydroxylation, beta-hydroxylation, and gamma-hydroxylation. The results of these studies on the activation and DNA binding of environmental nitrosamines will be important in assessing their possible roles in human cancer.